Fetal chromosomal aneuploidy diagnosis

ABSTRACT

The invention relates to prenatal detection methods using non-invasive techniques. In particular, it relates to prenatal diagnosis of a fetal chromosomal aneuploidy by detecting fetal and maternal nucleic acids in a maternal biological sample. More particularly, the invention applies multiplex PCR to amplify selected fractions of the respective chromosomes of maternal and fetal chromosomes. Respective amounts of suspected aneuploid chromosomal regions and reference chromosomes are determined from massive sequencing analysis followed by a statistical analysis to detect a particular aneuploidy.

FIELD OF THE INVENTION

This invention generally relates to the diagnostic testing of a fetalchromosomal aneuploidy by determining imbalances between differentnucleic acid sequences, and more particularly to the identification ofaneuploidy in chromosomes 13, 18, 21, X and/or Y via testing a maternalsample such as blood.

INTRODUCTION TO THE INVENTION

Aneuploidy refers to an abnormal number of chromosomes (or part ofchromosomes) that is a common cause of birth defects. In aneuploidy,genes can be present in three copies “trisomy” or in only one copy“monosomy”. These changes in chromosome number, resulting fromnondisjunction of chromosomes during meiosis, have dramatic effects onthe affected persons and result in well-known syndromes. The majority oftrisomies and monosomies are lethal to the fetus and cause spontaneousabortions or death immediately after birth. Some aneuploidies, however,are viable and result in syndromes. The most occurring aneuploidiesamong live births are chromosomes 21, 18, 13 trisomy's and a distortednumber of sex chromosomes. The most common autosomal aneuploidy thatinfants can survive with is trisomy 21 (Down syndrome), affecting 1 in800 births. Trisomy 18 (Edwards syndrome) affects 1 in 6,000 births, andtrisomy 13 (Patau syndrome) affects 1 in 10,000 births. Sex chromosomeaneuploidy (SCA) affects 1 in 400 newborns and is therefore, as a whole,more common than Down syndrome. While SCA include a variety ofabnormalities of the sex chromosomes, by far the most commonly occurringSCA is the deletion of chromosome X (45,X-Turner syndrome) or theaddition of an X or Y chromosome (47,XXY-Klinefelter syndrome, 47,XYY,47,XXX). Of these conditions, only Turner syndrome results in an easilyidentifiable physical phenotype. However, subtle language and learningdifficulties have been identified in most forms of SCA. The mostimportant risk factor for aneuploidy is maternal age since the majorityof children with aneuploidy are born to mothers over the age of 35, sothe prevalence is increasing as more women choose or need to delaychildbearing. Contemporary prenatal screening programs typically includethe common fetal chromosomal aneuploidies 21, 18 and 13. The risk of apregnancy is assessed by a number of means. For the chromosomalaneuploidies non-invasive screening tests based on ultrasonography andthe measurement of markers in maternal serum have been implemented toidentify high-risk pregnancies in the first 3 months of pregnancy (11-14weeks). The sonogram measures the fluid underneath the skin along theback of the baby's neck, called the nuchal translucency (NT). Thesonogram will also determine if the baby's nasal bone is present orabsent. A maternal blood sample is used to analyze two serum markerscalled free beta-human chorionic gonadotropin (hCG) and pregnancyassociated plasma protein-A (PAPP-A), which are found in the blood ofall pregnant women. In aneuploidy pregnancies there is extra fluidbehind the baby's neck and/or the hCG and PAPP-A results are higher orlower than average. Additionally, a baby's nasal bone may be absent insome pregnancies with a chromosome abnormality. Combining age-relatedrisk with the NT measurement, nasal bone data, and blood markers providea risk figure for Down syndrome and one risk figure for trisomy 13 ortrisomy 18. The first trimester screen's detection rate is approximately90% with a 5% false positive rate for pregnancies in which the baby hasDown syndrome, and is somewhat higher for pregnancies with trisomy 13 ortrisomy 18. A nuchal translucency sonogram can be performed withoutmeasuring hCG and PAPP-A. In this case, however, the aneuploidydetection rate is reduced to about 70%.

Prenatal diagnosis is an integral part of obstetric practice. To performa genetic diagnosis prenatally, genetic material from the unborn fetusis required. Conventionally, fetal DNA is sampled by invasive proceduressuch as amniocentesis or chorionic villus sampling. These procedures areassociated with a risk of miscarriage of respectively 0.5 and 1-2%.Hence, it is routine to reserve the invasive diagnostic procedures forpregnancies estimated to be at high aneuploidy risk which representabout 5-10% of all women screened for aneuploidy risk. Given theprocedure related risks of conventional prenatal diagnosis, it would beideal if genetic analysis of the fetus could be performednon-invasively. To perform non-invasive prenatal diagnosis, a source offetal genetic material without harming the fetus is therefore required.A major breakthrough to this end was reported by Lo et al. (1997)¹ andWO98/39474 describing the existence of free floating fetal DNA inmaternal plasma. They subsequently showed that fetal-derived DNAcontributed ˜10% of the free-floating DNA in maternal plasma. Fetal DNAcan be detected in maternal plasma just weeks after conception and israpidly cleared from maternal plasma and disappears within hours afterdelivery. As a result, free floating fetal DNA in maternal plasma is apromising source of fetal genetic material for the development of anon-invasive prenatal test. However, fetal DNA represents only a minorfraction of the total free floating DNA in plasma with the remainingportion of DNA contributed by the mother, mainly derived from maternalwhite blood cells.

Given the enormous potential, several non-invasive methodologies foraneuploidy detection have been described in the last decade. One methodis to focus on the analysis of nucleic acid molecules that arefetal-specific in maternal plasma and hence overcome the interferencecaused by the background maternal DNA. One could target the detection ofplacental expressed mRNA or placenta-specific epigenetic signaturesoriginating from the chromosome of interest. In a series of developmentssince 2000, the basis for plasma RNA as a prenatal diagnostic tool hasbeen established. Poon et al. (2000)² showed that mRNA transcribed fromthe Y chromosome could be detected in the plasma of women carrying malefetuses. Later, it was shown that the placenta is a major source offetal-derived RNA in maternal plasma using human placental lactogen mRNAand mRNA coding for the beta subunit of human chorionic gonadotrophin asexamples³.

In 2007, a placental-specific mRNA, transcribed from a gene located onchromosome 21, PLAC4, was identified using a microarray-based approachand was shown to be detectable in maternal plasma and cleared followingdelivery of the fetus⁴. To determine the dosage of chromosome 21 usingPLAC4 mRNA in maternal plasma the RNA-SNP allelic ratio approach wasused. This method is based on the presence of a SNP in the coding regionof the PLAC4 gene. If a fetus is heterozygous for this SNP, it possessestwo alleles that are distinguishable by DNA sequence. If the fetus iseuploid, the ratio of these two SNP alleles is 1:1. Conversely, if thefetus has trisomy 21, then the RNA-SNP allelic ratio would become 1:2 or2:1. Lo et al. (2007)⁴ demonstrated that this strategy could be appliedto non-invasively determine the chromosome 21 trisomy status of a fetus.Similarly, the RNA-SNP approach was also applied for the non-invasivedetection of trisomy 18 through the analysis of the allelic ratio ofSERPINB2 mRNA⁵.

The main limitation of the RNA-SNP allelic ratio approach, however, isthat only fetuses heterozygous for the analyzed SNP can be successfullydiagnosed. For example, with the use of the single SNP in PLAC4,approximately 45% of fetuses are expected to be heterozygous and thusdiagnosable using this approach. Consequently, several markers areneeded for full diagnostic coverage. To this end, a number ofinvestigators have described new polymorphic SNP markers that can beanalyzed using this approach. One preliminary report describes tenmarkers with a combined heterozygosity rate that covers up to 95% of theUS general population⁶. The evaluation of these markers in large-scaleclinical trials is expected over the next few years.

Placenta-specific epigenetic signatures, such as DNA methylation,originating from the fetal chromosome of interest have also beeninvestigated. As tissues in the body have different gene expressionprofiles, the methylation status of certain genes also exhibitstissue-specific patterns. Evidence shows that fetal DNA in maternalplasma originates from the placenta and that the maternal DNA backgroundis derived from maternal blood cells. Therefore, one way to developepigenetic fetal DNA markers is to identify genes whose methylationstatus differs between placental tissues and maternal blood cells. Chimet al. (2005)⁷ studied the methylation profile of the SERPINB5 (maspin)promoter and showed that it was hypomethylated in placental tissues buthypermethylated in maternal blood cells. Using methylation specific PCR,the placental-derived hypomethylated SERPINB5 could be detected anddistinguished from the maternally derived hypermethylated molecules inmaternal plasma. This made SERPINB5, located on chromosome 18, the firstuniversal circulating fetal DNA marker that could be used for allpregnancies regardless of fetal gender and genotype. Since the SERPINB5gene is located on chromosome 18, it allowed the development of astrategy that is analogous to the RNA-SNP allelic ratio approach, theso-called epigenetic allelic ratio approach. Thus, if a fetus isheterozygous for an SNP located in the promoter region of SERPINB5,measuring the ratio of the SNP alleles in the hypomethylated version ofthe gene, allows ascertainment of the fetus's trisomy 18 status.

However, methylation-specific PCR requires the use of a bisulphiteconversion, which alters unmethylated cytosines to uracil nucleotides.But, bisulphite conversion degrades up to 95% of the DNA molecules in asample and therefore substantially reduces the amount of fetal DNA in amaternal plasma sample and may result in false-negative detection.Consequently, researchers developed fetal epigenetic markers that couldbe detected in maternal plasma without the need for bisulphiteconversion. To this end, Chan et al. (2006)⁸ used the promoter ofRASSF1, located on chromosome 3, which is hypermethylated in placentaltissues but hypomethylated in maternal blood cells. Consequently, thehypomethylated RASSF1 sequences derived from the maternal blood cellscan be removed from maternal plasma using methylation sensitiverestriction enzyme digestion. Indeed, after restriction enzymedigestion, fetal RASSF1 sequences could be detected in maternal plasmabefore delivery but completely disappeared from maternal plasma within24h after delivery. Chan et al. (2006)⁹ used the differentialmethylation pattern of the RASSF1 promoter as the positive control forfetal DNA detection in a non-invasive prenatal fetal rhesus D bloodgroup typing for 54 early-gestation RhD-negative women.

The RNA-SNP allelic ratio approach and the DNA methylation approachtarget subsets of nucleic acid molecules present in maternal plasma in amolecular fashion. An alternative is using physical methods that resultin the relative enrichment of fetal DNA present in the maternal plasma.

Recently⁹, it was shown that the length of free floating fetal DNA inthe maternal plasma is ˜20 bp shorter than the maternally derivedfree-floating DNA. Therefore, size fractioning methods such as gelelectrophoresis allow size-fractionation of plasma DNA and enrichment ofthe shorter, fetal DNA fragments. This approach has been usedsuccessfully to enrich for free floating fetal DNA. While this approachhas been shown empirically to be useful for the qualitative detection ofdisease causing mutations, for example those causing beta-thalassemia,it is yet unknown whether the degree of enrichment might be sufficientfor fetal chromosomal aneuploidy detection requiring quantitativemeasurement of chromosome dosage. Dhallan et al. (2006)¹⁰ reportedanother approach for the enrichment of fetal DNA in maternal plasma.They hypothesized that a significant portion of maternal derivedfree-floating DNA in maternal plasma is released by maternal white bloodcells following phlebotomy. Therefore it was proposed that if maternalnucleated blood cells could be fixed, using formaldehyde, then thisdilution of fetal DNA in maternal plasma could be avoided. Dhallan¹⁰demonstrated the benefit of this approach for the noninvasive prenataldiagnosis of trisomy 21 showing a mean proportion fetal DNA of 34% in anexperiment comprising 60 pregnant women. However, the beneficial effectsof formaldehyde treatment could not be replicated by several othergroups.

The above-mentioned approaches are based on the assumption that the lowfractional concentration of fetal DNA in maternal plasma makes itchallenging to pursue the direct detection of fetal chromosomalaneuploidies. This is based on the limited precision of conventionalmethods for circulating fetal DNA detection, for example by real-timePCR.

The recent availability of single molecule counting techniques allowsdetection of fetal aneuploidy without the need to restrict the analysisto fetal-specific nucleic acids in maternal plasma. Digital PCR andmassively parallel sequencing are both single molecule counting methods,which allow the quantification of nucleic acids by counting moleculesand have superior analytical precision compared to conventional PCRbased detection methods. Digital PCR refers to the performance ofmultiple PCRs in parallel in which each PCR typically contains either asingle or no target molecule. Through the counting of the number ofpositive reactions at the end of amplification it is possible todetermine the number of input target molecules. Thus, they can preciselyquantify small increments in the total (maternal vs. fetal) amount ofDNA molecules derived from the aneuploid chromosome. Indeed, Lo et al.(2007)¹¹ demonstrated that the aneuploidy status detection is possibleeven when the trisomic DNA is present as a minor (10%) fraction. Thelower the fetal DNA concentration, the smaller the expected increment inthe amount of aneuploidy chromosome DNA. For digital PCR, quantitativeprecision improves with increasing number of PCR analyses performed. Loet al. (2007)¹¹ showed that accurate fetal trisomy 21 detection in amaternal plasma sample containing 25% fetal DNA requires about 8000digital PCRs to be performed, requiring the use of automated platformsin the clinical setting. Such automated platforms using microfluidicsare available (e.g. Fluidigm) but are expensive. Several groupsdemonstrated that non-invasive detection of fetal trisomy 21 could beachieved with the use of massively parallel, or next-generation,sequencing (e.g. WO2009/013496). Massively parallel sequencers allowanalysis of nucleotide sequences of millions to billions of DNAmolecules in each run. Therefore, in addition to the identity, afrequency distribution of the DNA molecules in the analyzed sample canbe obtained. Since free floating DNA in maternal plasma is fragmented innature it can be used directly to identify the chromosomal origin ofeach DNA molecule and determine the proportion of molecules derived froma potentially aneuploid chromosome. Several groups showed that theproportion of chromosome 21 DNA molecules in plasma of women pregnantwith a trisomy 21 fetus was elevated compared with that of euploidpregnancies. This approach was highly accurate for the direct detectionof fetal trisomy 21 from maternal plasma among small cohorts ofpregnancies.

Recently, two clinical validation studies were performed applying theabove-described method. In one study 449 samples were analyzed of which39 were trisomic for chromosome 21¹². A second study analyzed bloodsamples from 1014 at risk pregnancies collected in 13 US cliniclocations before they underwent an invasive prenatal procedure¹³. Ofthese 119 samples underwent massively parallel DNA sequencing.Fifty-three sequenced samples were classified correctly as having anabnormal fetal karyotype. Both clinical validation studies showedexcellent sensitivity and specificity. These data demonstrate thatplasma DNA sequencing is a viable method for noninvasive detection offetal trisomy 21 and warrants clinical validation in larger multicenterstudy.

On the other hand, it has been shown that the measurement of theproportional amounts of sequences derived from chromosomes with higheror lower GC contents then chromosome 21 was not as robust. Therefore,the measurements for chromosomes 18 and 13 are less precise and sufferfrom quantitative bias using trisomy 21 protocols. Thus, to achievereliable non-invasive detection of trisomy 18 and trisomy 13, sequencingand data analysis protocols that are less susceptible to the chromosomalGC content effects need to be developed and further validated. A recentstudy partially solved the above problem using a non-repeat maskedreference genome and a bioinformatics approach to correct GC contentbias in the sequencing data¹⁴. Using this approach all trisomy 13fetuses (25 out of 25) were detected at a specificity of 98.9% and 92%(34 out of 37) of the trisomy 18 fetuses at 98.0% specificity. Thesedata indicate that with appropriate bioinformatics analysis, noninvasiveprenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNAsequencing is not as reliable as trisomy 21.

In addition, the cost of massively parallel sequencing is high and thethroughput is low. Only a handful of cases can be analyzed per run,which takes several days. Further work is needed to develop morecost-effective protocols with higher throughput.

Recently, target enrichment was used to obtain a more efficient andcost-effective massive parallel sequencing approach¹⁵. This studyinvestigated the applicability in enriching selected genomic regionsfrom plasma DNA and the quantitative performance of this approach. Theexperiment showed that the mean sequence coverage of the enrichedsamples was ˜200-fold higher than that of the non-enriched samples andmore importantly that maternal and fetal DNA molecules were enrichedevenly. Furthermore, by using SNP data the authors were able to showthat the coverage of fetus-specific alleles within the targeted regionincreased from 3.5% to 95.9%. Overall, targeted sequencing of maternalplasma DNA allows efficient and unbiased detection of fetal alleles andis a powerful method for measuring the proportion of fetal DNA in amaternal plasma sample. Based on this single scientific paper targetenrichment shows great promise since it can reduce the sequencing costsubstantially. At the same time it requires an extra, enrichment stepthat will add an extra cost to the final test and also will delay thetest since a typical enrichment protocol takes about 24-36 hours tocomplete.

SUMMARY OF THE INVENTION

The present invention provides a non-invasive diagnostic DNA test foraneuploidy detection of chromosomes 21 and/or 18 and/or 13 and/or Xand/or Y by combining multiplex PCR based amplification of specific DNAsequences (i.e. targets) which contain at least one SNP in combinationwith sequencing technologies.

In another aspect the invention provides a non-invasive diagnostic DNAtest for aneuploidy detection of chromosomes 21 and 18 and 13 and X andY by combining multiplex PCR based amplification of specific DNAsequences (i.e. targets) which contain at least one SNP in combinationwith sequencing technologies.

Briefly, the present invention is directed to a method of differentialdetection of a predetermined set of target sequences in a mixture ofmaternal and fetal genetic material. Thus, the methods and materialsdescribed herein apply techniques for analyzing numerous nucleic acidscontained in a biological sample (preferably serum or plasma) containingfree floating DNA which is a mixture of DNA from both the mother and thefetus, and allowing detection of statistically significant differencebetween euploid and triploid fetuses. In contrast to the current massiveparallel sequence methods, based on whole genome or enriched samples,which do not achieve a sufficient sensitivity and specificity, inparticular, for chromosome 13, the present invention provides anon-invasive diagnostic assay with a specificity and sensitivity closeto 100% (respectively 99.99% specificity and 99.5% sensitivity) for thesimultaneous detection of chromosome 13, 18, 21, X and Y aneuploidies.Without limiting the invention to a particular theory or explanation,one reason why multiplex-PCR was not considered before in thedevelopment of non-invasive diagnostic aneuploidy tests is the presenceof high GC-rich regions particularly in chromosome 13. Yet anotherreason is that the use of multiplex-PCR was discouraged by one of theleading inventors (i.e. Dennis Lo) in US2010/0112590. Indeed, in thelatter application on paragraphs 116-117 it is recommended to applylocus-independent assays rather than locus-dependent assays such as forexample the targeted amplification carried out by the methods of thepresent invention.

Thus in one aspect the invention provides a method for determining thepresence or absence of fetal aneuploidy in a biological samplecomprising fetal and maternal nucleic acids present in free floating DNAfrom said maternal biological sample, amplifying a selected set oftarget DNA sequences in a quantitative (i.e. amplifying the template DNAsuch that the amplified DNA is reproducing the original template DNAratios) multiplex PCR reaction, conducting DNA sequencing of saidamplified selected set of target DNA sequences to determine the sequenceof said DNA sequences, using the obtained sequence data to compare anamount of amplified sequences derived from at least one first chromosomein said mixture of maternal and fetal DNA to an amount of amplified DNAsequences derived from at least one second chromosome in said mixture ofmaternal and fetal DNA, wherein said at least one first chromosome ispresumed to be euploid in the fetus, wherein said at least one secondchromosome is suspected to be aneuploid in the fetus, therebydetermining the presence or absence of said fetal aneuploidy.

In another aspect the invention provides a method for determining thepresence or absence of fetal aneuploidy in a biological samplecomprising fetal and maternal nucleic acids (such as free floating DNA)from said maternal biological sample, amplifying a selected set oftarget DNA sequences in a quantitative multiplex PCR reaction whereineach amplified DNA sequence comprises at least one SNP which isconsidered informative in case the pregnant female is heterozygous forthis SNP, conducting DNA sequencing of said amplified selected set oftarget DNA sequences to determine the sequence of said DNA sequences,using the obtained sequence data to compare an amount of amplifiedsequences which carry an informative SNP derived from at least one firstchromosome in said mixture of maternal and fetal derived DNA to anamount of amplified DNA sequences which carry an informative SNP derivedfrom at least one second chromosome in said mixture of maternal andfetal derived DNA, wherein said at least one first chromosome ispresumed to be euploid in the fetus, wherein said at least one secondchromosome is suspected to be aneuploid in the fetus, therebydetermining the presence or absence of said fetal aneuploidy and/ordetermining in said determined DNA sequences the allelic ratios of theinformative SNPs wherein a distorted allelic ration is indicative forthe presence of a fetal chromosomal aneuploidy in said pregnant female.

FIGURE LEGENDS

FIG. 1:

Dosage Quotients (DQ) of trisomic fetus when compared to euploid fetus.The grey shaded area indicates the expected percentages of fetal DNA.

FIG. 2:

Number of SNPs needed to gent minimally a given number of informativeSNPs, plotted per Minor Allele Frequency (MAF). The calculations aredone for a minimal probability of 99%.

FIG. 3: plot of expected vs. observed normalized read counts forchromosome 21 in a Down syndrome (trisomy 21) DNA samples (square) and 4euploid DNA samples (circles).

FIG. 4: plot of expected vs. observed normalized read counts for two ATPsamples (representing 20% trisomy 21) DNA samples (squares) and 4euploid DNA samples (circles).

FIG. 5: schematic representation of first multiplex PCR reaction of theMASTR assays procedure. Reverse and forward primers are ampliconspecific primers. Tag1 and Tag2 are universal sequencing that are usedin the second PCR reaction of the MASTR assay procedure to incorporate

FIG. 6: schematic representation of the second PCR reaction of the MASTRprocedure. In this step the MID sequences (barcodes) and A and Badaptors (for 454 emulsion PCR) are incorporated in the resultingamplicons from the first PCR reaction.

DETAILED DESCRIPTION OF THE INVENTION

The prior art has shown the feasibility of massive parallel sequencingas an analysis platform for free floating DNA based aneuploidy testing.However, current protocols result in expensive and low throughput testswhen used as a molecular diagnostic tool. The main reason for this isthe fact that current tests are based on genome wide sequencing of freefloating DNA resulting in the production of huge sequencing datasets ofwhich only a small fraction (˜5%) is used to determine the ploidy statusof the fetus. With this genome wide approach it is obligatory to use asubstantial part of the capacity of a massive parallel sequencerresulting in sequencing of a limited number of individuals per run,which takes several days to complete. Furthermore, huge sequencingdatasets are generated per individual that hamper efficient data storageand analysis.

The present invention offers a solution for this problem by using amultiplex-PCR based approach to amplify a number of selected chromosomalregions. Selected chromosomal regions are amplified in a multiplex PCRreaction from one or more chromosomes which are presumed to be aneuploidand selected set of chromosomal regions are amplified, preferably in thesame multiplex PCR reaction, from one or more chromosomes which arepresumed to be euploid. Chromosomes which are presumed to be euploid areherein further designated as a ‘reference chromosome’.

Accordingly the present invention provides in a first embodiment amethod for the detection of a fetal chromosomal aneuploidy in a pregnantfemale comprising i) receiving a biological sample from said pregnantfemale, ii) preparing nucleic acids from said biological sample, iii)amplifying a selected set of target DNA sequences in a quantitativemultiplex PCR reaction wherein at least one amplified DNA sequencecomprises at least one SNP which is considered informative if thepregnant female is heterozygous for this SNP, iv) sequencing of theamplified target DNA sequences and v) calculating the sum of read countsfor all amplified DNA sequences of a suspected chromosomal aneuploidyfollowed by normalization, against the sum of read counts for allamplified DNA sequences of a reference chromosome to determine bystatistical methods a set score indicative for the presence of a fetalchromosomal aneuploidy and/or determining the allelic ratios of theinformative SNPs wherein a distorted allelic ratio is indicative for thepresence of a fetal chromosomal aneuploidy in said pregnant female.

The term “biological sample” as used herein refers to any sample that istaken from a subject (e.g. such as a pregnant female or a pregnantwoman) and contains one or more nucleic acid molecule(s) of interest.

Accordingly a biological sample comprises for example blood, sputum,urine, cerebrospinal fluid (CSF), tears, plasma, serum, saliva ortranscervical lavage fluid.

The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA) and a polymer thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues The term nucleic acid is used interchangeably withgene, cDNA, mRNA, small noncoding RNA, micro RNA (miRNA),Piwi-interacting RNA, and short hairpin RNA (shRNA) encoded by a gene orlocus.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons). The term“reaction” as used herein refers to any process involving a chemical,enzymatic, or physical action that is indicative of the presence orabsence of a particular polynucleotide sequence of interest. An exampleof a “reaction” is an amplification reaction such as a polymerase chainreaction (PCR), preferably a multiplex PCR reaction. Another example ofa “reaction” is a sequencing reaction, either by synthesis or byligation. The term “clinically relevant nucleic acid sequence” as usedherein can refer to a polynucleotide sequence corresponding to a segmentof a larger genomic sequence whose potential imbalance is being testedor to the larger genomic sequence itself. Examples include chromosome18, 13, 21, X and Y. Yet other examples include mutated geneticsequences or genetic polymorphisms or copy number variations that afetus may inherit from one or both of its parents. The term “backgroundnucleic acid sequence” as used herein may refer to nucleic acidsequences originating from the mother or originating from the chromosomenot tested for aneuploidy in a particular analysis.

The term “free-floating DNA” is DNA which is derived from genomic DNA,free-floating DNA is in fact degraded genomic DNA and occurs in theextra-cellular space. As such free-floating DNA can be isolated frombody fluids (e.g. serum, plasma, sputum). The term “quantitative data”as used herein means data that are obtained from one or more reactionsand that provide one or more numerical values. The term “parameter” asused herein means a numerical value that characterizes a quantitativedata set and/or a numerical relationship between quantitative data sets.For example, a ratio (or function of a ratio) between a first amount ofa first nucleic acid sequence and a second amount of a second nucleicacid sequence is a parameter.

The term “cutoff value” as used herein means a numerical value whosevalue is used to arbitrate between two or more states (e.g. diseased andnon-diseased) of classification for a biological sample. For example, ifa parameter is greater than the cutoff value, a first classification ofthe quantitative data is made (e.g. diseased state); or if the parameteris less than the cutoff value, a different classification of thequantitative data is made (e.g. non-diseased state).

The term “imbalance” as used herein means any significant deviation asdefined by at least one cutoff value in a quantity of the clinicallyrelevant nucleic acid sequence from a reference quantity.

The term “chromosomal aneuploidy” as used herein means a variation inthe quantitative amount of a chromosome from that of a diploid genome.The variation may be a gain or a loss. It may involve the whole of onechromosome or a region of a chromosome. Examples of chromosomalaneuploidies are derived from chromosome 13, 18, 21, X and Y.

The term “random sequencing” as used herein refers to sequencing wherebythe nucleic acid fragments sequenced have not been specificallyidentified or targeted before the sequencing procedure.Sequence-specific primers to target specific gene loci are not requiredwhen random sequencing is applied. The pools of nucleic acids sequencedvary from sample to sample and even from analysis to analysis for thesame sample. In random sequencing the identities of the sequencednucleic acids are only revealed from the sequencing output generated incontrast to sequencing of multiplex-PCR amplified nucleotide sequences.

Embodiments of this invention provide methods, systems, and apparatusfor determining whether an increase or decrease (diseased state) of aclinically-relevant chromosomal region exists compared to a non-diseasedstate. This determination may be done by using a parameter of an amountof a clinically-relevant chromosomal region in relation to othernon-clinically-relevant chromosomal regions (background regions) withina biological sample. Nucleic acid molecules of the biological sample aresequenced, such that a fraction of the genome is sequenced, and theamount may be determined from results of the sequencing. One or morecutoff values are chosen for determining whether a change compared to areference quantity exists (i.e. an imbalance), for example, with regardsto the ratio of amounts of two chromosomal regions (or sets of regions).

The change detected in the reference quantity may be any deviation(upwards or downwards) in the relation of the clinically-relevantnucleic acid sequence to the other non-clinically-relevant sequences.Thus, the reference state may be any ratio or other quantity (e.g. otherthan a 1-1 correspondence), and a measured state signifying a change maybe any ratio or other quantity that differs from the reference quantityas determined by the one or more cutoff values.

The clinically relevant chromosomal region (also called a clinicallyrelevant nucleic acid sequence or suspected aneuploid chromosome orchromosomal region) and the background nucleic acid sequence may comefrom a first type of cells and from one or more second types of cells.For example, fetal nucleic acid sequences originating fromfetal/placental cells are present in a biological sample, such asmaternal plasma, which contains a background of maternal nucleic acidsequences originating from maternal cells. Preferentially, maternal andfetal nucleic acid sequences are derived from free-floating DNA. In oneembodiment, the cutoff value is determined based at least in part on apercentage of the first type of cells in a biological sample. Note thepercentage of fetal sequences in a sample may be determined by anyfetal-derived loci and not limited to measuring the clinically-relevantnucleic acid sequences.

In another embodiment the methods of the invention use cell (e.g. bloodcells) stabilizing chemicals in the preparation of the nucleic acidspresent in the biological sample which is received from the pregnantfemale. Indeed, one of the major technical challenges in usingfree-floating fetal DNA from maternal blood is the low fraction of fetalDNA present in the sample. This fraction is typically between 10 and 20%in the first trimester of pregnancy (week 11-14), which corresponds withthe stage where an aneuploidy DNA test is best performed. This lowfraction of fetal DNA is even for molecular counting methods challengingwith respect to the sensitivity and specificity of the test. Thereforeit is important to maximize the ratio fetal/maternal free floating DNA.The present invention provides different solutions for this problem.

In a particular embodiment the disruption of nucleated blood cells isprevented during the collection, storage or transport of the biologicalmaterial, in particular a maternal blood sample prior to plasmaisolation. This is important to prevent dilution of fetal DNA resultingin a decreased ratio fetal/maternal free floating DNA. Severalcommercial cell stabilizing blood collection tubes are available whichstabilize blood cells for at least 14 days at room temperature allowingconvenient sample collection, transport and storage (available forexample at www.streck.com).

In yet another particular embodiment a size fractionation is used in themethods of the invention to prepare maternal and fetal nucleic acids.

Indeed, the prior art shows that fetal and maternal free-floating DNAhave different size distributions. Free floating fetal DNA is generally20 bp shorter than the maternal free floating DNA and this observationcan be used to further enrich the free-floating fetal DNA fraction ifthis smaller sized fraction is specifically separated from the maternalfraction. One way to accomplish this is by means of gel electrophoresis.In a particular embodiment, a gel electrophoresis basedsize-fractionating device is used as marketed by Sage Science(www.sagescience.com). This device is a fully automated system enablingtight size selection and a high recovery rate. Furthermore, iteliminates the cross contamination risk completely since all samples areseparated from each other during the whole size fractionation process.

In a particular embodiment the amplified DNA sequences obtained in thequantitative multiplex PCR reaction in the methods of the invention havea size between 80 and 140 base pairs.

In view of the size distributions of the fetal and maternal freefloating DNA populations it is essential to keep the amplified DNAsequence lengths below 140 bp to ensure efficient amplification of theshorter fetal free-floating DNA fraction.

Preferred amplified DNA sequence lengths are between 80 and 140basepairs.

In yet another embodiment the amplified DNA sequences obtained in onesingle multiplex PCR reaction are between 30 and 60.

In yet another embodiment the amplified DNA sequences obtained in onesingle multiplex PCR reaction are between 60 and 80.

In yet another embodiment the amplified DNA sequences obtained in onesingle multiplex PCR reaction are between 70 and 80.

Preferably only one quantitative multiplex PCR reaction is applied topractice the methods of the invention.

In yet another embodiment the GC-content of the target DNA sequences(i.e. the DNA sequences which are amplified with the quantitativemultiplex PCR reaction) is between 30% and 70%. Our experimental datapoint out that a range of 40%-60% GC is optimal for a close to 100%sensitivity and specificity of the methods of the invention.

An essential step in the methods of the present invention is thesequencing of the amplified target DNA sequences. As a high number ofsequencing reads, in the order of hundred thousand to millions or evenpossibly hundreds of millions or billions can theoretically be generatedfrom each sample in each run, the resultant sequenced reads form arepresentative profile of the mix of nucleic acid species in theoriginal biological sample. However, the person skilled in the art wouldknow how many runs to perform based on the stage of pregnancy (which iscorrelated with the amount of free-floating fetal DNA in the biologicalsample) and based on the origin of the biological sample derived from apregnant female. The most important aspect is that a high degree ofstatistical confidence is obtained. In order to improve statisticalconfidence, it is preferable to perform a large number of reads,preferably between 10.000 and 100.000 or more reads, depending on thepercentage of fetal DNA present in the mixture. A commonly used measureof statistical significance when a highly significant result is desiredis p<0.01, i.e. a 99% confidence interval based on a chi-square ort-test.

In a preferred embodiment massive parallel sequencing methods are used.In particular embodiments, the sequencing is done using massivelyparallel sequencing. Massively parallel sequencing, such as for exampleon the 454 platform (Roche) (Margulies, M. et al. 2005 Nature 437,376-380), Illumina Genome Analyzer (or Solexa platform) or SOLID System(Applied Biosystems) or the Helicos True Single Molecule DNA sequencingtechnology (Harris T D et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRT™) technology of Pacific Biosciences, andnanopore sequencing (Soni G V and Meller A. 2007 Clin Chem 53:1996-2001), allow the sequencing of many nucleic acid molecules isolatedfrom a specimen at high orders of multiplexing in a parallel fashion.Each of these platforms sequences clonally expanded or evennon-amplified single molecules of nucleic acid fragments.

An important advantage of the limited set of amplified nucleotidesequences which is generated by the methods of the present invention isthat emerging low cost and lower capacity massive parallel sequencerscan be used such as the 454 junior (Roche), PGM (Life Technologies) orMiSeq (Illumine). The combination of the methods of the invention andthe low end sequencers results in a fast turnaround time per test sincethese platforms typically take only a few hours per sequencing run. Inaddition, the lower cost is also an important improvement over themethods used in the prior art.

In a particular embodiment the massive parallel sequencing data areanalyzed by calculating the sum of read counts for all amplified DNAsequences of a suspected chromosomal aneuploidy (e.g. all amplified DNAsequences derived from chromosome 21 and/or chromosome 13 and/orchromosome 18 and/or chromosome X and/or chromosome Y) are counted (i.e.the number of times a specific amplified chromosomal sequence is presentin the biological sample). The sum of read counts for the amplified DNAsequences derived from a particular suspected aneuploid chromosome (e.g.chromosome 13 or 18 or 21 or X or Y) is then normalized against the sumof read counts for the amplified DNA sequences derived from a referencechromosome (i.e. a chromosome for which no aneuploidy is reported).Thus, the multiplex PCR allows the calculation of dosage quotients (DQs)by comparing (target region read count, i.e. the suspected aneuploidychromosome or chromosomal region)/(control region read count, i.e. thereference chromosome or chromosomal region) ratios between the pregnantfemale and the fetus. The DQs in function of the percentage fetal DNA isdepicted in FIG. 1.

An essential element of the methods of the present invention is that theamplified target DNA sequences are reflecting identical ratios of theamounts of maternal and fetal free floating nucleic acids in thebiological sample and hence the methods require quantitativeamplification. Based on multiplex PCR assays and the PCR conditions usedto amplify samples (limited number of cycles) we previously showed thattemplate DNA is amplified quantitatively¹⁶. If there is a normaldistribution between the two read counts then a score (e.g. a Z-score ora dosage quotient) is obtained. A Z-score of 1 means that there is noaneuploidy for the suspected aneuploidy chromosome. A Z-score higherthan 1, preferentially higher than 2, more preferentially higher than 3,is an indication for the presence of an aneuploidy of the chromosome. Itis understood that Z-scores are determined for all the suspectedaneuploidy chromosomes for which a selected set of target DNA sequencesare obtained by the methods of the invention. The normalization and thecalculation of the Z-score is assisted by the use of statisticalmethods. Useful statistical methods which can be used in the context ofthe present invention include Bayesian-type likelihood method,sequential probability ratio testing (SPRT), false discovery, confidenceinterval and receiver operating characteristic (ROC).

In yet another particular embodiment the massive parallel sequencingdata of the amplified target DNA sequences are analyzed based on thedetermination of the allelic ratios of the informative SNPs wherein adistorted ratio is indicative for the presence of a fetal chromosomalaneuploidy in the pregnant female. The allelic ratio is distorted forinformative SNPs on aneuploid chromosomes. This distortion can bemeasured when the mother is heterozygous for a given SNP (referredherein as “informative SNP”). Therefore, sequence analysis of the MASTRassay will result in a number of informative SNPs that can be used todetermine the fetal ploidy status on top of the fetal ploidy statusdetermination by molecular counting as described above. FIG. 2 shows theresult of a calculation of the number of informative SNPs with a 99%probability provided a minor allele frequency (MAF) between 0.25 and0.50. Based on this calculation it is depicted in FIG. 2 that with aminimal MAF of 0.25 at least 7 informative SNPs are present in a set of35 amplified target DNA sequences, while 10 informative SNPs areidentified for SNPs with a MAF of 0.50.

In yet another particular embodiment the massive parallel sequencingdata of the amplified target DNA sequences are analyzed based on thedetermination of the allelic ratios of the informative SNPs wherein adistorted ratio is indicative for the presence of a fetal chromosomalaneuploidy in the pregnant female in combination with calculating thesum of read counts for all amplified DNA sequences of a suspectedchromosomal aneuploidy (e.g. all amplified DNA sequences derived fromchromosome 21 and/or chromosome 13 and/or chromosome 18 and/orchromosome X and/or chromosome Y) are counted (i.e. the number of timesa specific amplified chromosomal sequence is present in the biologicalsample).

In yet another embodiment based on carrying out the methods of theinvention a classification of whether a fetal chromosomal aneuploidyexists for one or more suspected aneuploid chromosomes determined. Inone embodiment, the classification is a definitive yes or no. In yetanother embodiment, a classification may be unclassifiable or uncertain.In yet another embodiment, the classification may be a score that is tobe interpreted at a later date, for example, by a medical doctor.

In particular embodiments the bioinformatics, computational andstatistical approaches used to determine if a biological sample obtainedfrom a pregnant woman conceived with an aneuploid chromosome orchromosomal region or euploid fetus could be compiled into a computerprogram product used to determine parameters from the sequencing output.The operation of the computer program would involve the determining of aquantitative amount from the potentially aneuploid chromosome as well asamount(s) from one or more of the other chromosomes. A parameter wouldbe determined and compared with appropriate cut-off values to determineif a fetal chromosomal aneuploidy exists for the potentially aneuploidchromosome.

In yet another embodiment the invention provides a diagnostic kit forcarrying out the method of the invention. Such a diagnostic kitcomprises at least a set of primers to amplify target maternal andtarget fetal nucleic acids wherein these target nucleic acids arederived from chromosome 13 and/or chromosome 18 and/or chromosome 21and/or chromosome X and/or chromosome Y. Preferentially the kitcomprises primers for amplifying target nucleic acids derived fromchromosomes 13, 18, 21, X and Y. In addition, the diagnostic kitcomprises a set of primers which are able to identify target DNAsequences of a reference chromosome or a reference chromosomal part. Itis understood that such a reference chromosome or part thereof is aneuploid chromosome. Euploid refers to the normal number of chromosomes.Other reagents which can optionally be included in the diagnostic kitare instructions and a polymerase and buffers to carry out thequantitative polymerase multiplex PCR reaction.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

1. Prenatal Diagnosis of Fetal Trisomy 21

The DNA samples used in the present examples are samples prepared bymixing a diploid DNA sample derived from a female (representing thematernal DNA) with either a male DNA sample sample euploid forchromosome 21 (referred to as artificial euploid pregnancy or AEP) orwith a male DNA sample triploid for chromosome 21 (referred to asartificial trisomy pregnancy or ATP). Each artificial sample wascomprised of a mixture of 80% maternal DNA and 20% of male DNA. Inaddition, included in the analysis was a DNA sample derived from a Downsyndrome individual, having 3 copies of chromosome 21.

Measurements were performed on 4 AEP samples, 2 ATP samples and 1 Downsyndrome DNA sample. For each measurement, approximately 50 ng of DNAwas used in a standard 2-step MASTR assay PCR amplification procedure(see Materials and Methods). The fetal chromosome 21 MASTR assay iscomprised of 20 primer pairs derived from chromosome 21 and 10 primerpairs derived from chromosome 18. The resulting amplicons from eachMASTR amplified individual DNA sample contained a specific barcode. Theresulting barcoded amplicons of each DNA sample were equimolarly mixedand subjected to the 454 junior emulsion PCR protocol as described bythe manufacturer. After emulsion PCR, beads were isolated and loaded ona 454 junior according to the manufacturer's protocol. A total of two454 junior runs were performed in order to obtain sufficient reads toreach a per amplicon coverage between 300 and 500.

Since the Down syndrome DNA sample contains 3 chromosome 21 copies, itshould provide 50% more chromosome 21 reads then the AEP samples. Tocalculate this, the following calculation steps were performed on theDown sample and on the AEP samples:

-   -   (i) Read counts for each chromosome 18 and 21 amplicon was        divided by the total number of chromosome 18 derived read counts    -   (ii) For each chromosome 18 and 21 amplicon, the average read        count over the different AEP samples was calculated    -   (iii) For each chromosome 18 and 21 amplicon, (i) was divided by        (ii)    -   (iv) For each chromosome and each sample, the average value        of (iii) was calculated    -   (v) The observed normalized ratio chromosome21/chromosome18 was        calculated by dividing averages calculated under (iv) per AEP        and ATP

FIG. 3 shows a plot of the observed (calculated as above) and expected(i.e. theoretical values) number of read counts for chromosome 21amplicons of the Down DNA sample.

These data show that a clear distinction can be made between a normal,euploid DNA sample and a trisomy (i.e. Down syndrome), chromosome 21 DNAsample.

To evaluate the feasibility to distinguish between an euploid sample(represented by the AEP artificial samples) and an artificial chromosome21 aneuploidy sample containing 20% chromosome 21 trisomy derived DNA,the above calculations were performed on the ATP samples relative to theAEP samples.

A presence of 20% of trisomy DNA in the ATP samples should result in a10% increase in chromosome 21 amplicon read count compared to the AEPsamples. Indeed using the above calculations, FIG. 4 shows a cleardistinction between the AEP and ATP samples reflecting an approximately10% increase in chromosome 21 in the two ATP samples.

Material and Methods

1. Primer Sequences Used in the Examples

TABLE 1 list of 30 primer pairs composing thechromosome 21 aneuploidy detection MASTR assay Amplicon Chrom Forw RevNITT_089 chr18 AAGACTCGGCAGCATCTCCATTTG GCGATCGTCACTGTTCTCCAGAGAGAGTTAGCTTGACTTTGG TGGTATTAGGAAGGTTTGGT NITT_092 chr18AAGACTCGGCAGCATCTCCACACT GCGATCGTCACTGTTCTCCAGTGG TTCTCCTAACACCCTTGGGTGTCCTTAGGGGTCT NITT_096 chr18 AAGACTCGGCAGCATCTCCATCAGGCGATCGTCACTGTTCTCCACTCA CACTCCCTCCATGA AAGAAATGGAAGAGAATACAAAA NITT_097chr18 AAGACTCGGCAGCATCTCCACCTG GCGATCGTCACTGTTCTCCAGGCA CATCTTGACACAGTCGTCCAGGAGGAGAAAA NITT_093 chr18 AAGACTCGGCAGCATCTCCAGGATGCGATCGTCACTGTTCTCCAGAAG GGTCACAGTGGGTCA AGGGGAGAAGTAGAGGTTAAA NITT_085chr18 AAGACTCGGCAGCATCTCCATAAG GCGATCGTCACTGTTCTCCAGAGGCAAACAGCAGCACAAAA GAATCTGTAATCCACATGA NITT_094 chr18AAGACTCGGCAGCATCTCCACCAG GCGATCGTCACTGTTCTCCACTCC AGTGGAATTGCTGAGACTTCTCTTTCTTCTTCTTCTAAGC NITT_084 chr18 AAGACTCGGCAGCATCTCCATGCAGCGATCGTCACTGTTCTCCATTAA GATGGAGGACATCGT ATTTGCTCTTGGTATACTTCTTGNITT_083 chr18 AAGACTCGGCAGCATCTCCATGGT GCGATCGTCACTGTTCTCCATCACCCAGTTGGAGGGTCT AGATGACATGGAAAATAAGC NITT_091 chr18AAGACTCGGCAGCATCTCCATAAA GCGATCGTCACTGTTCTCCACCCA AGTGCCTTTGAACTCTGACTATGTGAAATCGCATAGTT NITT_050 chr21 AAGACTCGGCAGCATCTCCACATCGCGATCGTCACTGTTCTCCACAAC CAGGACCTACCATCTTG GCTGGCATTCAAAA NITT_010 chr21AAGACTCGGCAGCATCTCCACCTT GCGATCGTCACTGTTCTCCAGTGT CTCACTCACCTCTTTCTTGGCAGAGGAGAGACATGA NITT_011 chr21 AAGACTCGGCAGCATCTCCATGTGGCGATCGTCACTGTTCTCCATATG TGTGTGTTCTCTACCTTGG AGTAGGTGTCTGGTGTATGAAAANITT_047 chr21 AAGACTCGGCAGCATCTCCAGCAA GCGATCGTCACTGTTCTCCATCAGATCTGGTACTGGGTATGA ATAGTATGGATAAAGGCAATGA NITT_006 chr21AAGACTCGGCAGCATCTCCACAAT GCGATCGTCACTGTTCTCCACATT AATCAGACTTTGCCTTGGAAGGGTCTTAGGGTGGTAAA NITT_049 chr21 AAGACTCGGCAGCATCTCCATCCTGCGATCGTCACTGTTCTCCAGAAG GTTGGGGAAATTGG ATAGAGTTTCTCCTGCATCA NITT_017chr21 AAGACTCGGCAGCATCTCCAGGAA GCGATCGTCACTGTTCTCCACAGC CAGGTGCACACATCAACTGTCCAGCACTTG NITT_007 chr21 AAGACTCGGCAGCATCTCCACAGCGCGATCGTCACTGTTCTCCAGTCT TGTAACCTGCTGAGAAAA TAATTCTGCTCAGGAAAAGCNITT_009 chr21 AAGACTCGGCAGCATCTCCAGAAC GCGATCGTCACTGTTCTCCATTGAAGCATTCCTCCTCCTAGT ACCATAAATGTCAGCTCTTG NITT_070 chr21AAGACTCGGCAGCATCTCCACCTC GCGATCGTCACTGTTCTCCATTCC ACATGTCTGTGCATTAAAACTCTTCACATTCTGCTC NITT_071 chr21 AAGACTCGGCAGCATCTCCAGACAGCGATCGTCACTGTTCTCCACTCT CAACATCAGAGGCAATCT TCAAACAGAGAAAACTTAGATGANITT_016 chr21 AAGACTCGGCAGCATCTCCATCAG GCGATCGTCACTGTTCTCCACAGAGGTAGAGAATCAGAATTGG GATCAACCGGAGAAGTAAA NITT_076 chr21AAGACTCGGCAGCATCTCCACCAC GCGATCGTCACTGTTCTCCAGTTC GGATCCACTGCATATCTGTAAGTGAAAGCATCCTAAA NITT_057 chr21 AAGACTCGGCAGCATCTCCATCTGGCGATCGTCACTGTTCTCCAGAGG GTCTAAATAAAGTCTTCACATCA TAGGAAATGCACCATCANITT_020 chr21 AAGACTCGGCAGCATCTCCACAGA GCGATCGTCACTGTTCTCCAGAAAGGCCATGCCAGTAGT GTCTGGGAGGTTGAAGC NITT_039 chr21AAGACTCGGCAGCATCTCCATGCC GCGATCGTCACTGTTCTCCACACA ATCAGAACCCGTAAACAGAAGCACAGGAAAATC NITT_059 chr21 AAGACTCGGCAGCATCTCCACCTTGCGATCGTCACTGTTCTCCATAGT CTCTGCCTCCATTCTAGT GTCCGATAATGAAGAACAGTAAANITT_053 chr21 AAGACTCGGCAGCATCTCCATGGC GCGATCGTCACTGTTCTCCACTGATAAGCACATACCCTTAAA CACAAATGAAGGCAAAA NITT_044 chr21AAGACTCGGCAGCATCTCCATCTC GCGATCGTCACTGTTCTCCAGAAC CATTCCTTCTGCTCTTAGTTCACTCTGGAAGCAATGA NITT_072 chr21 AAGACTCGGCAGCATCTCCAGAAAGCGATCGTCACTGTTCTCCAGAAC GCTGGGCGTATTGG ATTCTGAACATCTGGAATGA

2. MASTR Assay Principle

Primerpairs were first tested in simplex PCR reactions on 20 ng ofgenomic DNA using 10 pmol per primer; the other parameters were equal tothose of the multiplex PCR. The multiplex PCR reactions were performedon 50 ng genomic DNA in a 25-ml reaction containing Titanium™ Taq PCRbuffer (Clontech, Palo Alto, Calif.) with a final concentration of 0.25mM for each dNTP (Invitrogen, Carlsbad, Calif.) and a total of 0.125 mlof Titanium™ Taq DNA Polymerase (Clontech). Primer concentrations wereoptimized and varied between 0.05 pmol/ml and 0.2 pmol/ml finalconcentration.

The final multiplex assay (MASTR assay) was used to amplify all DNAsamples. The first PCR reaction was performed on 50 ng of DNA withfollowing settings: initial sample denaturation 10 min at 95° C.followed by 20 cycles each consisting of: 45 sec at 95° C., 45 sec at60° C. and 2 min at 68° C. ending with a final extension step of 10 minof 72° C. (see FIG. 5).

The resulting PCR fragments were 1000 times diluted followed by a secondPCR step to incorporate the individual barcode. The PCR conditions ofthis step are identical to the conditions of the first PCR step (seeFIG. 6).

The resulting barcoded amplicons are equimolarly mix and used in anemulsion PCR reaction as described by the manufacturer (Rochediagnostics).

REFERENCES

¹Lo Y, Corbetta N, Chamberlain P, Rai V, Sargent I, Redman C, andWainscoat J (1997) Presence of fetal DNA in maternal plasma and serum.The Lancet 350: 485-487

²Poon L, Leung T, Lau T, Lo Y (2000) Presence of fetal RNA in maternalplasma. Clin Chem 46: 1832-1834

³Ng E, Tsui N, Lau T, Leung T, Chiu R, Panesar N, et al. (2003) mRNA ofplacental origin is readily detectable in maternal plasma. PNAS 100:4748-4753

⁴Lo Y, Tsui N, Chiu R, Lau T, Leung T, Heung M, et al. (2007) Plasmaplacental RNA allelic ratio permits noninvasive prenatal chromosomalaneuploidy detection. Nat Med 13:218-23

⁵Tsui N,2, Wong B, Leung T, Lau T, Chiu R and Lo Y (2009) Non-invasiveprenatal detection of fetal trisomy 18 by RNA-SNP allelic ratio analysisusing maternal plasma SERPINB2 mRNA: a feasibility study. Prenat Diagn29: 1031-1037

⁶Yang Y, Ding J, Lee M, Loria 0. Mohsenian F, Tang M, et al. (2008)Identification of mRNA-SNP markers for a noninvasive prenatal trisomy 21(T21) test. Prenat Diagn 2008: 28-S12

⁷Chim S, Tong Y, Chiu R, Lau T, Leung T, Chan L, et al. (2005) Detectionof the placental epigenetic signature of the maspin gene in maternalplasma. PNAS 102: 14753-14758

⁸Chan K, Ding C, Gerovassili A, Yeung S, Chiu R, Leung T et al. (2006)Hypermethylated RASSF1A in Maternal Plasma: A Universal Fetal DNA Markerthat Improves the Reliability of Noninvasive Prenatal Diagnosis. ClinChem 52: 2211-2218

⁹Lo D, Chan A, Sun H, Chen E, Jiang P, Lun F et al. (2010) MaternalPlasma DNA Sequencing Reveals the Genome-Wide Genetic and MutationalProfile of the Fetus. Sci Transl Med 2: 6

¹⁰Dhallan R, Guo X, Emche S, Damewood M, Bayliss P , Cronin M et al.(2007) A non-invasive test for prenatal diagnosis based on fetal DNApresent in maternal blood: a preliminary study. Lancet 369: 474-481

¹¹Lo D, Lun F, Chan A, Tsui Y, Chong C, Lau T, et al. (2007) Digital PCRfor the molecular detection of fetal chromosomal aneuploidy. PNAS104:13116-131121

¹²Ehrich M, Deciu C, Zwiefelhofer T; Tynan J, Cagasan L, Tim R et al.(2011) Noninvasive detection of fetal trisomy 21 by sequencing of DNA inmaternal blood: a study in a clinical setting. Am J Obstet Gynecol204:205.e1-11

¹³Sehnert A, Rhees B, Comstock D, de Feo E, Heilek G,1 Burke J and RavalP (2011) Optimal Detection of Fetal Chromosomal Abnormalities byMassively Parallel DNA Sequencing of Cell-Free Fetal DNA from MaternalBlood. Clin Chem 57: 1042-1047

¹⁴Chen E, Chiu R, Sun H, Akolekar R, Chan A, Leung T et al. (2011)Noninvasive Prenatal Diagnosis of Fetal Trisomy 18 and Trisomy 13 byMaternal Plasma DNA Sequencing. PLoS ONE 6: e21791

¹⁵Liao G, Lun F, Zheng Y, Chan A, Leung T, Lau T et al. (2011) TargetedMassively Parallel Sequencing of Maternal Plasma DNA Permits Efficientand Unbiased Detection of Fetal Alleles. Clin Chem 57: 92-101

1-11. (canceled)
 12. A method for analyzing DNA sequencing reads,comprising sequencing amplified target DNA sequences from at least onequantitative multiplex PCR reaction, wherein the amplified target DNAsequences are from a biological sample of a pregnant female and compriseone or more chromosomal regions suspected of being aneuploidy and one ormore chromosomal regions of a reference chromosome; and calculating thesum of read counts for all amplified target DNA sequences of thesuspected chromosomal aneuploidy followed by normalization, against thesum of read counts for all amplified DNA sequences of the referencechromosome to determine by statistical methods a set score indicativefor the presence of a fetal chromosomal aneuploidy.
 13. The method ofclaim 12, wherein said fetal chromosomal aneuploidy is chromosome 13and/or chromosome 18 and/or chromosome 21 and/or chromosome X and/orchromosome Y.
 14. The method of claim 12, wherein said biological sampleis maternal blood, plasma, urine, cerebrospinal fluid, serum, saliva oris transcervical lavage fluid.
 15. The method of claim 12, wherein theamplified target DNA sequences have a size between 30-180 base pairs.16. The method of claim 12, wherein the GC content of the amplifiedtarget DNA sequences is between 20 and 70%.
 17. The method of claim 12,wherein the amplified target DNA sequences are obtained in a singlemultiplex PCR reaction.
 18. The method of claim 17, wherein in saidsingle multiplex PCR reaction more than 40 amplified DNA sequences areobtained.
 19. The method of claim 17, wherein in said single multiplexPCR reaction more than 60 amplified DNA sequences are obtained.
 20. Akit comprising primers capable of amplifying target nucleic acidsequences from chromosomes 13, 18, and/or 21, and primers capable ofamplifying a reference chromosome, wherein each primer has a tag orbarcode.
 21. The kit of claim 20, wherein the kit comprises primerscapable amplifying target nucleic acid sequences from chromosome 13, andprimers capable of amplifying a reference chromosome.
 22. The kit ofclaim 20, wherein the kit comprises primers capable amplifying targetnucleic acid sequences from chromosome 18, and primers capable ofamplifying a reference chromosome.
 23. The kit of claim 20, wherein thekit comprises primers capable amplifying target nucleic acid sequencesfrom chromosome 21, and primers capable of amplifying a referencechromosome.
 24. The kit of claim 20, wherein the kit comprises primerscapable amplifying target nucleic acid sequences from chromosome 13, 18,and 21, and primers capable of amplifying a reference chromosome. 25.The kit of claim 20, further comprising a polymerase and buffer.
 26. Thekit of claim 21, further comprising a polymerase and buffer.
 27. The kitof claim 22, further comprising a polymerase and buffer.
 28. The kit ofclaim 23, further comprising a polymerase and buffer.
 29. The kit ofclaim 24, further comprising a polymerase and buffer.